In recent years, epigenetics has been recognized as being involved in various biological phenomena, and research concerning epigenetic analysis has been actively conducted. In particular, it has been revealed that aberrant methylation of DNA is deeply involved in canceration, and this fact has been attracting attention. As a characteristic epigenetic aberration in cancer cells, there is known aberrant DNA methylation of CpG islands in promoter regions of some genes. The CpG island refers to a region in which a two-nucleotide sequence of cytosine (C) and guanine (G) linked through a phosphodiester bond (p) occurs at a high frequency, and the CpG island is often present in a promoter region upstream of a gene. The aberrant DNA methylation of the CpG island is involved in carcinogenesis through, for example, inactivation of a tumor suppressor gene. Canceration is induced by inactivation of various tumor suppressor genes such as CDKN2A, CDH1, MLH1, RB, BRCA1, TSLC1, and RUNX3, through aberrant methylation of CpG islands present in their promoter regions. When DNA is methylated, the methylated DNA is replicated at the time of cell division and passed on to daughter cells. Accordingly, when a tumor suppressor gene is inactivated by aberrant methylation, the tumor suppressor gene continues to be in the inactivated state.
As an already established analysis method for methylated DNA, there is known a method involving utilizing a hydrogen sulfite (bisulfite) reaction. This method is the most generally used method for analysis of methylated DNA. When single-stranded DNA is treated with the hydrogen sulfite, cytosine is converted to uracil through sulfonation, hydrolytic deamination, and desulfonation. On the other hand, methylated cytosine has an extremely low reaction rate in the sulfonation that occurs first, and hence remains as methylated cytosine in a reaction time of the hydrogen sulfite treatment to be actually performed. Therefore, when a polymerase chain reaction (PCR) is performed using the DNA treated with the hydrogen sulfite, methylated cytosine remains as cytosine, whereas unmethylated cytosine is amplified as thymine in place of uracil. The base difference between cytosine and thymine, which occurs in a sequence of a PCR amplification product, is utilized to analyze a methylation status. Generally used methods that adopt the foregoing as their basic principle are a methylation-specific PCR (MSP) method described in Patent Document 1 and Non Patent Document 1, and a combined bisulfite restriction analysis (COBRA) method described in Non Patent Documents 2 and 3.
The MSP method is a method involving, after the hydrogen sulfite treatment of DNA, performing PCR amplification with a methylated sequence-specific primer and a unmethylated sequence-specific primer, and agarose gel electrophoresis in the stated order, and determining a DNA methylation status of a region of interest based on the presence or absence of amplification products obtained with both the primers. The COBRA method is a method involving, after the hydrogen sulfite treatment of DNA, performing PCR amplification with a primer common to methylated DNA and unmethylated DNA, treatment with a restriction enzyme that recognizes a site of a sequence difference between methylated DNA and unmethylated DNA, and agarose gel electrophoresis in the stated order, and determining a DNAmethylation status of a region of interest based on the presence or absence of a restriction enzyme-treated fragment. Both the methods are still widely used methods because the methods allow quantitative analysis of methylated DNA without any special apparatus. However, there has been a problem in that those methods take time and effort in the analysis owing to the utilization of the electrophoresis method.
Meanwhile, for separation assay of biopolymers such as nucleic acids, proteins, and polysaccharides in the fields of biochemistry, medicine, and the like, ion-exchange chromatography is generally used as a method that allows accurate detection to be performed simply and within a short period of time. The ion-exchange chromatography is a method of separating a substance as a measurement object through the utilization of an electrostatic interaction occurring between an ion-exchange group of a column packing material and an ionic group in the substance as the measurement object. One type of this method is based on anion exchange and another is based on cation exchange. The anion-exchange chromatography allows an anionic substance to be separated through the use of a column packing material having a cationic functional group as the ion-exchange group. In addition, the cation-exchange chromatography allows a cationic substance to be separated through the use of a column packing material having an anionic functional group as the ion-exchange group.
In separation of PCR amplification products of nucleic acids using the ion-exchange chromatography, use is generally made of the anion-exchange chromatography that performs the separation by utilizing negative charge of phosphate contained in a nucleic acid molecule. The cationic functional group of the column packing material in the anion-exchange chromatography is classified into a weak cationic group such as a diethylaminoethyl group, and a strong cationic group such as a quaternary ammonium group. Columns packed with column packing materials having those cationic functional groups as the ion-exchange groups are already commercially available, and used in various fields of research.
In addition, one of the inventors of the present invention developed, as a column packing material for ion-exchange chromatography, a column packing material having both a strong cationic group and a weak cationic group as cationic functional groups of the column packing material, and reported separation assay of a single-base difference between 20-mer unmethylated synthetic oligonucleotides by ion-exchange chromatography using a column packed with this packing material (Patent Document 2).